1. Volume 9, Issue 6, Pages 819-830 (December 2005)
Phosphorylation by Double-Time/CKIε and CKIα Targets Cubitus Interruptus for Slimb/β- TRCP-Mediated Proteolytic Processing 
Jianhang Jia, Lei Zhang, Qing Zhang, Chao Tong, Bing Wang, Fajian Hou, Kazuhito Amanai, Jin Jiang 
Developmental Cell 
Volume 9, Issue 6, Pages (December 2005)
DOI: /j.devcel
Copyright © 2005 Elsevier Inc. Terms and Conditions

2. Figure 1 DBT/CKIε Is Required for Ci Processing
(A and B) A wild-type wing (A) or a wing containing a clone of cells expressing a dominant-negative form of DBT/CKIε (DN-DBT) with act>CD2>Gal4 (B). An anteriorly situated DN-DBT-expressing clone induces duplication of wing tissues (arrow, B).
(C–C″) A late third instar wing disc expressing UAS-DN-DBT with act>CD2>Gal4 was immunostained to show Ci155 accumulation (detected by the Ci antibody 2A) and CD2 expression. DN-DBT-expressing clones were marked by the lack of CD2 expression. Anterior DN-DBT-expressing cells accumulate high levels of Ci155 cell autonomously (arrow). All the wing discs shown in this study are oriented with anterior to the left and ventral at the top.
(D–D″) A wing disc expressing UAS-DN-DBT with act>CD2>Gal4 was immunostained to show Ci155 accumulation and dpp-lacZ expression. Anterior DN-DBT-expressing clones accumulate high levels of Ci155 and ectopically express dpp-lacZ (arrows).
(E–G) Wing discs of the genotypes wild-type (E), dco3/dcole88 (F), and dcoP103/dcole88 (G) were immunostained with Ci 2A antibody. Ci155 was accumulated in A compartment cells of dcoP103/dcole88 wing disc but not in dco3/dcole88 wing disc.
(H–H″) High-magnification view of a wing disc containing dcole88 clones and immunostained to show Ci155 accumulation and CD2 expression. dcole88 clones are marked by the lack of CD2 staining (arrow).
(I–I″) High-magnification view of a wing disc containing dcole88 clones generated with a wing-specific flipase (MS1096/UAS-FLP) and immunostained to show Ci155 accumulation and dpp-lacZ expression. Anterior dcole88 cells marked by the lack of CD2 expression (not shown) accumulate Ci155 but fail to activate dpp-lacZ (arrows).
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

3. Figure 2 CKIα Acts in Conjunction with CKIε to Promote Ci Processing
(A–C′) A wild-type wing disc (A and A′) and wing discs expressing CKIα RNAi construct CRL (B and B′) or CRS (C and C′) with MS1096 were immunostained to show Ci155 accumulation and dpp-lacZ expression. High levels of CRL expression in anterior-dorsal cells result in Ci155 accumulation and ectopic dpp-lacZ expression (arrows, B and B′). Cells expressing CRL at reduced levels accumulate Ci155 but do not ectopically express dpp-lacZ (arrowheads, B and B′). Wing discs expressing CRS accumulated modest levels of Ci155 in a dorsal compartment of cells but failed to ectopically activate dpp-lacZ (arrows, C and C′).
(D and D′) A dco3/dcole88 wing disc expressing CRS with MS1096 Gal4 was immunostained to show Ci155 accumulation and dpp-lacZ expression. dco3/dcole88 mutant cells expressing high levels of CRS accumulate Ci155 to high levels and ectopically express dpp-lacZ (arrows).
(E–G) Wing discs expressing DBT/CKIε kinase domain (CKIεKD) alone (E) or in conjunction with CRL (F) or CRS (G) with MS1096 were immunostained with an anti-CKIε N terminus antibody.
(H–J) Wing discs expressing Flag-tagged CKIα (Fg-CKIα) alone (H) or together with either CRL (I) or CRS (J) were immunostained with an anti-Flag antibody.
(K and K′) A wing disc coexpressing CRL and CKIεKD was immunostained to show Ci155 accumulation and dpp-lacZ expression. Ci155 accumulation and ectopic dpp-lacZ expression induced by CRL was suppressed by CKIεKD (arrows).
(L) A wing disc coexpressing CRS and CKIεKD was immunostained to show Ci155 accumulation. Ci155 accumulation induced by CRS was suppressed by CKIεKD (arrow).
(M–O) Wing discs expressing mC∗ alone (M) or in conjunction with DN-CKIε (N) or CRL (O) with MS1096 were stained to show Ci155 accumulation.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

4. Figure 3 PKA- and GSK3-Primed Phosphorylation of Ci by CKI
(A) A schematic drawing of Ci155. The Zn finger DNA binding domain and dCBP binding domain are indicated as blue and red boxes, respectively. The vertical bars indicate the five PKA consensus sites. Sequence surrounding the first three PKA sites and its alignment to the corresponding regions in Gli proteins are shown underneath. PKA and GSK3 sites are color coded in red and green, respectively. PKA- and GSK3-primed CKI sites are shown in blue and orange, respectively.
(B) A list of phosphorylation-deficient Ci mutants with corresponding amino acid substitutions is indicated.
(C) PKA-primed phosphorylation of Ci by CKI. GST-Ci fusion proteins containing wild-type (CiWT, lanes 1–3) or mutant (CiSA16) Ci fragment (amino acids 812–908) were incubated with recombinant PKAc or CKIδ in the presence of γ-32P-ATP, or treated with CKIδ in the presence of γ-32P-ATP and PKA inhibitor PKI following a prior incubation with PKA in the presence of unlabeled ATP (indicated by an asterisk).
(D) GSK3-primed phosphorylation of Ci by CKI at S855. GST-Ci fusion proteins containing wild-type or mutant Ci fragments from amino acids 846–869 were either treated with PKAc alone (lanes 5 and 6) or with PKAc and GSK3β in the presence of cold ATP, followed by incubation with CKIδ in the presence of γ-32P-ATP, PKI, and GSK3 inhibitor LiCl (lanes 1–4).
(E) Late third instar wild-type (WT) wing discs (lanes 1, 2, and 5) or wing discs expressing CRL with MS1096 (lanes 3 and 4) were treated with (lanes 2–5) or without (lane 1) 50 μM MG132 and 12.5 nM OA, separated on 7.5% SDS-PAGE, and Western blotted with anti-Ci (2A) antibody, which recognizes Ci155. The numbers of discs used for each lane are 15 for lanes 1, 2, and 5; 6 for lane 3; and 10 for lane 4. Of note, fewer CRL-expressing discs were used, as they are larger than wild-type discs and accumulate high levels of Ci155. The open arrow indicates the position of fast-migrating, hypophosphorylated or unphosphorylated species of Ci155, whereas the filled arrow indicates the slow-migrating, hyperphosphorylated Ci155. Ci155 from CRL-expressing discs (lanes 3 and 4) exhibits increased mobility as compared with that from wild-type discs (lanes 2 and 5).
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

5. Figure 4 Mutating Individual CKI Sites Affects Ci Processing
Late third instar wing discs bearing smo3 clones and expressing HA-tagged wild-type or CKI site-deficient forms of Ci with MS1096 were stained to show the expression of hh-lacZ (red) and CD2 (green). smo3 clones were marked by the lack of CD2 expression. P compartment smo3 cells expressing HA-Ci block hh-lacZ expression (A and A′). P compartment smo3 clones expressing HA-CiSA34 (C and C′), HA-CiSA56 (D and D′), HA-CiSA36 (E and E′), or HA-CiSA855 (I and I′) partially inhibit hh-lacZ expression. P compartment smo3 clones expressing HA-CiSA16 (F and F′), HA-CiSA34G (G and G′), or HA-CiSA34C (H and H′) fail to inhibit hh-lacZ expression. P compartment smo3 cells expressing HA-Ci and CRL fail to block hh-lacZ expression (J and J′).
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

6. Figure 5 Binding of Slimb to Phosphorylated Ci
(A) GST-Ci was phosphorylated by the indicated kinases and then incubated with in vitro translated 35S-labeled full-length Slimb. Slimb bound to glutathione beads was precipitated by centrifugation and separated on SDS-PAGE, followed by autoradiography. The right panel is a protein gel showing GST-Ci. The arrow indicates the full-length protein, whereas the asterisks indicate the breakdown products.
(B) GST-Ci was phosphorylated by PKA, GSK3, and CKI, and then incubated with in vitro translated 35S labeled full-length (FL), N-terminal domain (N), or WD40 repeats (WD) of Slimb. Phosphorylated GST-Ci pulled down the full-length and WD repeats of Slimb but not the N-terminal Slimb fragment.
(C) GST, GST-CiWT, GST-Ci−PKA, and GST-CiSA16 were phosphorylated with PKA and CKI and then incubated with in vitro translated 35S-labeled full-length Slimb. Bound Slimb was separated on SDS-PAGE, followed by autoradiography. The protein gel (right) indicates equal amounts of individual GST fusion proteins were used.
(D) GST or GST-Ci fusion proteins containing the wild-type sequence (GST-CiWT) or the DSGXXS motif (GST-CiDSG) were phosphorylated by the indicated kinases, and incubated with in vitro translated 35S-labeled full-length Slimb. GST-CiDSG but not GST-CiWT pulled down a significant amount of Slimb after phosphorylation by PKA and GSK3.
(E) Alignment of the Slimb binding sites (boxed) present in Iκ-Bα and β-catenin, with the consensus sequence shown underneath.
(F) Sequence of the three phosphorylated clusters in wild-type Ci (CiWT), CiDSG, CiDAG, and CiDSGX2.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

7. Figure 6 The DSGXXS Motif Confers Ci Processing Independent of CKI
Late third instar wing discs bearing smo3 clones and expressing HA-CiDSG (A and B′), HA-CiDAG (D and D′), or HA-CiDSGX2 (C and C′) with MS1096 were immunostained to show the expression of hh-lacZ (red) and CD2 (green). smo3 clones were marked by the lack of CD2 expression. P compartment smo3 cells expressing HA-CiDSG (arrows, A, A′, B, and B′) or HA-CiDSGX2 (arrows, C and C′) blocked hh-lacZ expression. In contrast, P compartment smo3 cells expressing HA-CiDAG failed to inhibit hh-lacZ expression (arrows, D and D′). P compartment smo3 cells expressing HA-CiDSGX2 no longer inhibited hh-lacZ expression when PKA activity was blocked by coexpressing R∗ (arrows, E and E′). P compartment smo3 cells expressing HA-CiDSGX2 still inhibited hh-lacZ expression when CKI activity was blocked by coexpressing CRL (arrows, F and F′).
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions

8. Figure 7 A Model for Ci Processing
The full-length activator form of Ci (Ci155) is phosphorylated at multiple sites in its C-terminal half by PKA, which primes its further phosphorylation by GSK3 and CKI (CKIα and CKIε). GSK3 phosphorylation can prime Ci for further phosphorylation by CKI. Sequential phosphorylation by these three kinases creates three phosphorylation clusters that function as docking sites to recruit SCFSlimb, followed by proteasome-mediated proteolytic processing to generate a C-terminally truncated repressor form of Ci (Ci75). See text for detail.
Developmental Cell 2005 9, DOI: ( /j.devcel )
Copyright © 2005 Elsevier Inc. Terms and Conditions